Sexual transmission through mucosal surfaces has been the most common route of HIV-1 spread throughout the world. Although much attention has been focused on vaccine development for HIV-1, progress has been slow and there is an urgent need to find alternative approaches, such as topical microbicides, to target the spread of HIV-1. In our search for novel compounds active against HIV-1, we discovered that the cyclic antimicrobial peptides called retrocyclins were potent inhibitors of HIV-1 replication. We have synthesized and tested the anti-HIV-1 activity of nearly 120 analogs of retrocyclin, and determined that the lysine analog RC-101 was best suited for development as a topical microbicide. RC-101 was shown to be active against R5 and X4 HIV-1, and acts by preventing the initiation of the fusion complex by binding the heptad repeat region 2 on gp41, precluding six helix bundle formation. RC-101 also exhibited little to no cytotoxicity or induction of proinflammatory cytokines in organotypic cervicovaginal epithelia and cervical organ culture, did not hemagglutinate red blood cells, was remarkably stable, and could be formulated into thin films for intravaginal application. Pilot studies with film-formulated RC-101 in five pigtail macaques revealed that the peptide was safe, exhibited no appreciable toxicity, and was associated with cervicovaginal epithelial cells. Based on our findings, we have formed several hypotheses about RC-101: a) RC-101 is stable and bioavailable, thus will likely function to inhibit HIV-1 infection in the vaginal mucosa, b) RC-101 (an entry/fusion inhibitor) and CSIC (a non-nucleoside reverse transcriptase inhibitor being developed by Project 1) have different mechanisms of action and thus their activities will be complementary in preventing HIV transmission, and c) RC-101, formulated as a controlled-release product, can provide long-term protection against heterosexual HIV-1 transmission. With assistance from a multidisciplinary team, we will test these hypotheses by 1) determining the broad spectrum efficacy, stability in biologic fluids and ability to induce HIV-1 resistance of RC-101, alone and in combination with CSIC, and 2) evaluating silicone elastomer ring-formulated RC-101, alone and in combination with CSIC, in organotypic cervicovaginal tissues and following topical vaginal administration in monkeys. These studies involve a highly collaborative and iterative approach to develop RC-101 in combination with CSIC as a ring-formulated intravaginal topical microbicide.